Visualizing single-molecule conformational transition and binding dynamics of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) play crucial roles in cellular processes and hold promise as drug targets. However, the dynamic nature of IDPs remains poorly understood. Here, we construct a single-molecule electrical nanocircuit based on silicon nanowire field-effect transistors (SiNW-FETs) and functionalize it with an individual disordered c-Myc bHLH-LZ domain to enable label-free, in situ, and long-term measurements at the single-molecule level. We use the device to study c-Myc interaction with Max and/or small molecule inhibitors. We observe the self-folding/unfolding process of c-Myc and reveal its interaction mechanism with Max and inhibitors through ultrasensitive real-time monitoring. We capture a relatively stable encounter intermediate ensemble of c-Myc during its transition from the unbound state to the fully folded state. The c-Myc/Max and c-Myc/inhibitor dissociation constants derived are consistent with other ensemble experiments. These proof-of-concept results provide an understanding of the IDP-binding/folding mechanism and represent a promising nanotechnology for IDP conformation/interaction studies and drug discovery.

1. The agreement in the HRMS characterization between theoretical and observed MWs is suspiciously high. Such agreement might happen 5% of the time, but every molecule with a tiny difference in the submilli-amu seems too good to be true. I do not believe the authors have made the molecules they claim to have made and suggest that they use the checklist for characterization required for organic chemicals by organic chemistry journals (e.g., JOC). In other words, I'd like to see a fully assigned 1H and 13C NMR spectra and LC-MS to check purity, etc. Without such data, the reader cannot believe the results as a presented.
13. "n" in the manuscript has been clarified according to the comments from Reviewer #4. Please see Figs. 1-5 in the main text.
14. The descriptions about the LC46-inhibitor interaction have been added for a better understanding according to the comments from Reviewer #4. Please see Pages 13 and 15 in the main text.

Point-to-point response to the reviewers' comments (in blue)
Reviewer #1 (Remarks to the Author):

Comments:
This is a very interesting manuscript that describes the use of a silicon nano wire functionalized with a single molecule of an intrinsically disordered protein (c-Myc).
Through measurements of current, the authors study the conformational ensemble of c-Myc as well as its interaction with a binding partner (Max) and available inhibitors. The authors clearly demonstrate the sensitivity of this approach to probe different conformational ensembles of the free IDP, as well as its structural transitions in the presence of binding partner and inhibitors. Overall, I think the work provides an important contribution to the IDP field. I am in favor of publication in Nature Communications, if the following points can be addressed: 1) Page 6: The authors provide evidence for a single-LC46 functionalization by attaching FITC for fluorescence characterization. It was not clear how this method can unambiguously determine that the devices were modified with a single LC46 molecule, when the LC46 sequence contains six lysines. This aspect needs to be clarified, also in terms of the achievable resolution for the fluorescence characterization.

Response:
We thank the reviewer for the comment. In order to verify the single-LC46 functionalization with fluorescence methods, we introduced FITC as the fluorescent group. Since there are multiple lysine reactive sites in the LC46 sequence, we carried out the reaction at a low FITC concentration to reduce the reactivity. We utilize  and Supplementary Fig. 17). The device without FITC modification was also characterized using atomic force microscopy (AFM, Bruker AFM Dimension Icon). The AFM image (Supplementary Fig. 18) was generated at the ScanAsyst mode with a sampling rate of 1.00 Hz and 512 samples per line, which confirmed the presence of a single LC46 attached to the etched gap on the side of silicon nanowires.
2) Page 7: The authors assign the observed decrease in current to partially or transiently folded states of LC46 induced by positive charges coming closer to the SiNW. Would it be possible to provide some more evidence for this using additional experiments modulating the screening of charges (using different salt concentrations for example)?
The charge distribution of LC46 is almost uniform, meaning that the positively charged residues (K/R) are interspersed with negatively charged amino acids (D/E). Globally, LC46 has a charge of +3 (or only +2, if the NH at the N-terminus of the protein is not counted). On this basis, it is difficult to understand how this charge is the only parameter modulating the current. It would be important to understand in more detail the physical origin of the observed current changes, in order to provide evidence for a general applicability of the method to other IDP systems (with varying pIs and sequence compositions).

Response:
We thank the reviewer for the constructive comment. According to the Debye screening effect, the electrical double layer (EDL) formed in ionic solution around the surface of silicon nanowires screens the charge signals from the solution environment beyond the Debye length, including random collisions of charged ions and charged molecules (Nano Lett. 2012, 12, 5245;Nano Lett. 2015, 15, 2143 Some references to literature should be added to support the claim that LC46 is mostly disordered for example from NMR experiments or maybe small angle X-ray scattering.
Are such data available?

Response:
We thank the reviewer for the suggestion on. According to the NMR results using the δ2D method, the free c-Myc352-437 showed intrinsic disordered nature in contrast to the results of the ordered structure of the Myc-Max complex (Biochemistry 2019, 58, 3144;PLOS. Comput. Biol. 2013, 9, e1003249). In addition, the CD spectra (Sci. Rep. 2016, 6, 22298.) also showed that the apo state of c-Myc370-409 (6 amino acids shorter than LC46) was disordered.  (Fig. R2). Therefore, we did provide further discussion in the revised main text.    Supplementary Fig. 30). 7) It was not clear to which extent kinetic information can be extracted (koff and kon rates for the c-myc/max complex).

Response:
We thank this reviewer for the comment. By using the QuB software (Biophys. Rev. & Lett. 2013, 8, 191), the kinetic information can be extracted through the idealization of current trajectories of the LC46 (encounter state) association with DQ47 that allows stabilization of the fully folded LC46 in the heterodimer, as shown in Supplementary Fig. 32   Oure Revision: We have revised Fig. 1 as below. -What is the reason for using a DMSO-containing buffer for the experiments? Is that for comparability with the measurements using inhibitors? I am asking because some proteins may not be compatible with these conditions.

Fig. 1 | Schematic demonstration and characterization of a c-Myc-modified SiNW
Response: Thanks a lot for the questions and comments. The DMSO was employed to increase the solubility of LC46. A DMSO-containing buffer was used in previous research on c-Myc and its inhibitors and it was shown that low concentration of DMSO had minor effect on the protein structure (PLOS Comput. Biol. 2013, 9, e1003249;Sci. Rep. 2016, 6, 22298).
-Please add temperature to the experiments in Fig. 2a. We would like to take this opportunity to thank this reviewer for the precious time and important suggestions for us to improve the manuscript. We hope this reviewer will find this revised version satisfactory. Sincerely, The Authors ------End of Response to Reviewer #1------Reviewer #2 (Remarks to the Author): The manuscript describes the construction of a single-molecule electrical nanocircuit functionalised by the intrinsically disordered region of the cancer-associated protein c-Myc and its use to study conformational transitions in c-Myc either in its apo state or in response to binding to its binding partner Max as well as binding to a small molecule inhibitor. Overall, the manuscript is well written and the data figures are appropriate.
Major points: 1) A major conclusion of the manuscript relates to the potential identification of a prebinding state. However, the interpretation of the "additional" experimental signal as a 2) The authors only focus on induced fit as a model for explaining their experimental data and completely neglect conformational selection, which is a bit surprising giving previous evidence of the presence of a transient a-helical region in the IDR of c-Myc prior to binding.
Response: Thanks a lot for this suggestive comment. We apologize for our negligence on the conformation selection mechanism. Relevant discussions have been added to the revised manuscript. We agree with the reviewer that the presence of transient folded conformation of LC46, which we also captured in our experiments. We calculated the kobs as the number of occurrences per second of the fully folded binding complex. The As a result, we cannot conclude that LC46 either undergoes an induced-fit mechanism or a conformational selection mechanism. However, an intermediate state from free LC46 to the fully folded binding complex was detected. A relatively flat energy landscape was observed in the apo-LC46 experiments, where the most local minimums were unable to cause enough effective gating voltage. The presence of DQ47 and inhibitors gave rise to LC46 folding into several more stable conformational ensembles, which were captured by the SiNW-FET devices as the high frequent cluster signals.
These partially folded ensembles tended to form a relatively stable one, which was favorable for the formation of the fully folded binding complex.  The kobs can be fitted by Eq. R2 derived from a two-step induce-fit model. Red for pH 7.4 and dark red for pH 6.5.

Page 5 in the Supplementary
Page 6 in the main text: The materials and detailed methods are provided in the Supplementary Information (Supplementary Figs. 1-16).
2. The authors claim that only one protein is attached per SiNW-FET. However, they show this using a FITC labeling of the protein and then imaging to observe only one label. First, FITC labelling is not quantitative. The conditions could label a low percentage of proteins that are present. Second, FITC is readily photo-bleached, which again would lower the yield of active FITC labels present. The authors could use inliquid AFM to rigorously demonstrate single molecule attachment. Or they could moderate the manuscript's claims.
Response: Thanks for the suggestion. We agree with this reviewer on the unreliability of FITC labels because of the un-quantified factors and the low yield of active FITCs.
In order to further validate the single-LC46 modification, we conducted AFM experiments (Fig. R1) to verify the single-LC46 modification.
Our Revision: We have added Fig. R1 as Supplementary Fig. 18   We would like to take this opportunity to thank this reviewer very much for all the time involved and important suggestions for us to improve the manuscript. We hope this reviewer will find this revised version satisfactory. Sincerely, The Authors ------End of Response to Reviewer #3------ 1. What would be the difference between the binding-induced-folding described here and the induced-fit model?
Response: Thanks for the suggestive comment. We think that binding-induced-folding is the same as the induced-fit model for IDPs (Curr. Opin. Struct. Biol. 2009, 19, 31).
However, after conscientious consideration on the mechanism, we cannot be sure that LC46 undergoes an induced-fit mechanism or a conformational selection mechanism. Page 11 in the main text: Based on the concentration-dependent signal patterns, we propose an interaction hypothesis (Fig. 1d)

Page 16 in the main text: In addition, the competition experiments showed the presence of different encounter intermediate ensembles upon binding to different ligands, and again demonstrated the encounter-intermediate process for the Myc-Max
interaction.

Page 17 in the main text: An encounter intermediate mechanism for the Myc-Max interaction was also revealed by real-time electrical monitoring, including a folding transition among conformational intermediates at low Max concentrations and a binding equilibrium at high Max concentrations.
2. The device schematic image (Fig 1a) is not scaled, which may confuse readers. The authors need to provide more information on the dimension of the nanogap and the scale of LC46? If the nanogap is too large, DQ47/inhibitors could interact directly with SiNW, regardless of the presence or absence of LC46, leading to similar random telegraph signals. If the nanogap is tight, the LC46 would be confined in the nanogap, limiting its conformational motions and increasing its potential to be sticking to the SiNW or Su-8 if it is in a disordered, long chain-like state as depicted in Fig1a.    Fig. 3a originated from the interaction between LC46 and DQ47 ( Supplementary Fig. 26a). Fig. 4 Figs. 26b and 26c).

came from the interaction between LC46 and inhibitors (Supplementary
3. The authors need to explain the method used to control the attachment of a single protein. What was the yield? Also, was the attachment stable for the long-duration measurement? There could be dissociation, significant degradation, unfolding or misfolding during the long-term, temperature-dependent measurements. It is important to know how many devices and proteins were tested in the manuscript, and confirm that the signals are reproducible and the dwell times are with different binding partners and temperatures.
Response: Thanks a lot for the suggestions. The yield of the single-LC46 devices was ~10% on each chip. For each experiment, we at least tested three different devices to verify that the signals analyzed were reproducible. We agree with the reviewer's opinion about the stability of the attachment. The temperature and long-time measurement did have an impact on the device. When temperature reached above 45 ℃, the device experienced irreversible changes and the signals could not be detected.
However, the stability of the device can at least ensure us to obtain the data we needed.
The signals were reproducible according to Fig. R19. The dwell times were different at different temperature but similar when different devices were measured under the same condition. Our Revision: We have added Fig. R19 as Supplementary Fig. 31. The information about the yield and the stability of devices, reproducible signals has been added in the  Supplementary Fig. 31).  (Fig. R1) to verify the single-LC46 modification.
Our Revision: We have added Fig. R1 as Supplementary Fig. 18 1b and Supplementary Fig. 17). The device without FITC modification was also characterized using atomic force microscopy ( Supplementary Fig. 18) Only a single fluorescence spot was reconstructed on individual SiNW-FET devices ( Fig. 1b and Supplementary Fig. 17). The device without FITC modification was also characterized using atomic force microscopy (AFM, Bruker AFM Dimension Icon). The AFM image (Supplementary Fig. 18)   Page 6 in the main text: The materials and detailed methods are provided in the Supplementary Information (Supplementary Figs. 1-16).
where ε is the dielectric permittivity of the media, kB is Boltzmann's constant, T is the temperature, q is the electron charge, and c represents the ionic strength of the electrolyte (Nano Lett. 2012, 12, 5245.). Table R1 shows the Debye lengths of solutions in our experiments.
In addition, after the mutation, the properties of disordered peptide will change, further bringing about more complex changes, which is not the current focus of this work and can be further explored in the future.   Fig. R21 as Supplementary Fig. 20 not only from 1-dimensional motions of LC46, but from the changes of total effective gating caused by the global conformational changes of LC46. The changes of LC46 with total positive charge from a relatively extending conformation ensemble to a partially folded or compact binding conformation will lead to a change of the effective gating, therefore giving rise to the decrease of currents.
8. The authors need to provide more data or compelling arguments for the transient coiled and partially folded state (Fig. 1). Our Revision: We have added Fig. R18 as Supplementary Fig. 26 in the Supplementary Information. Relevant description has been added on Page 11 in the main text: Based on these concentration-dependent signal patterns, we propose an interaction hypothesis (Fig. 1d) Fig. 3c). As DQ47 concentration further increase, the equilibrium of the binding process would finally shift to the fully folded binding structure (100 nM -5 M DQ47 in Fig 3c).
9. (Page 9, lines 1-3). It is not obvious that 1* is a dynamic self-folding process? Why does the protein undergo a self-folding process at low and high temperatures? The authors need to provide a more convincing argument and experimental support for this.
Response: Thank this reviewer very much for the comments and suggestions. We apologize for our unclear explanations about state 1*. State 1* was a current state derived from the self-folding process, which occurred with the similar current level to state 1, but with different characteristic dwell time (as shown in Fig. 2c, two different peaks occurred in the dwell time distribution of the medium current level. the dominant one [dark green] was state 1, the other one was state 1*). After careful consideration about state 1*, we realized that the re-emerging state 1* at high temperatures might not be the same as that at lower temperature in spite of their similar current levels. As a result, we revised Figs. 2d and e as Fig. R22. State 1*LT exhibited a similar current level to state 1, but with longer dwell times, and disappear at 35 ℃.   11. Similar to the previous point, the authors need to provide details on the charges and charge distribution of DQ47, and they get close to the SiNW when bound? How does binding result in substantial changes in the conductance of the devices?

Response:
We thank this reviewer for the comments. The conformation of LC46 mainly contains a random coil structure with short and transient helical structures as revealed by previous studies (Sci. Rep. 2016, 6, 22298 andPLOS Comput. Biol. 2013, 9, e1003249). After binding to DQ47 to form a stable heterodimer (as indicated by the complex structure: PDB: 1NKP), LC46 folds into helix-loop-helix conformation which dramatically shortens its radius of gyration. The charge distribution of the well folded LC46-DQ47 complex was depicted in the following figure since DQ47 only could not influence the current changes. Thus, we think that the substantial changes in the conductance were mainly affected by charge distribution changes induced by the dramatic conformational changes.
Our Revision: We have added relevant descriptions about the conformational change and conductance changes in the main text.
Pages 10-11 in the main text: The gating voltage was mainly affected by charge distribution changes induced by the dramatic conformational changes.
12. How do the authors know that DQ47 induces induction of LC46 without direct interaction? Need more data or convincing arguments.
Response: Thanks for the comment. We apologize for our inaccurate description about the induction which led to misunderstanding. We considered that DQ47 did interact with LC46 before specific binding. Because of the disordered nature of DQ47 and LC46, DQ47 did not initially form a tightly specific binding complex structure with LC46, but a probable loose interaction system. LC46 undergoes folding caused by the interaction with DQ47 to form the encounter intermediate ensemble, further forming a tight complex structure.
Our Revision: We have revised the description about the interaction DQ47 on Page 11 in the main text: Based on these concentration-dependent signal patterns, we propose an interaction hypothesis (Fig. 1d) Fig. 3c). As DQ47 concentration further increase, the equilibrium of the binding process would finally shift to the fully folded binding structure (100 nM -5 M DQ47 in Fig 3c).
13. This manuscript shows multiple conformational populations of LC46, and thus it could be feasible to determine these conformational states via crystal structure analysis.
Are there any crystal structures of LC46 or LC46-DQ47 complexes available to support the findings in this manuscript?
Response: We thank this reviewer for the comments and suggestions. The CD spectra of Fig 2a in the literature (Sci. Rep. 2016, 6, 22298) showed that the apo state of c-Myc370-409 (6 amino acids shorter than LC46) was disordered and the NMR data of Fig   5 in the literature (Biochemistry 2019, 58, 3144) also demonstrated the intrinsic disordered nature of free c-Myc352-437. As a result, LC46 itself is disordered which precludes crystal structure determination. As LC46 and DQ47 can form stable complex, the corresponding crystal structure of Myc-Max heterodimer complex has been reported before (PDB: 1NKP).
14. The temperature dependence of intermediate states, binding, and dissociation constants could be further validated by FACS or fluoresces assaying experiments, which will provide stronger evidence.
Response: It is a good idea to use fluorescent experiments as demonstration for our results. We have tried very hard to carry out fluorescent experiments. Unfortunately, we were not successful in synthesizing the labeled LC46 due to low solubility. In addition, we are also worried that the attached fluorescent labels on the disordered peptides may affect their conformational properties. We will continue to try in future studies.
15. The LC46-inhibitor complexes show similar changes in current direction and amplitude (dI) as the LC46-DQ47 complex, indicating that both inhibitors have the same level of positive charges of DQ47. Why/how does the current drop for the LC46inhibitor complex? What are the charges for 10074 and 1205 and how the charges are determined? To verify the signal direction and amplitude, the authors could repeat these experiments using synthetic compounds with additional positive or negative charges.

Response:
We thank the reviewer for the comments and suggestions. In fact, neither 10074-A4 nor PKUMDL-YC-1205 have positive or negative charges. As DQ47 could induce the fully folded (more condensed) conformation of LC46, the similar changes in current dI indicated that these small molecules also have the potential to induce extended-to-condensed conformational changes of LC46. In the case of DQ47, LC46 folds into a more compact structure (fully folded complex). Coupled with additional positive charge carried by DQ47, the current level of LC46-DQ47 complex is smaller than that of LC46-inhibitor complex as shown in the competition experiments ( Fig. 5 and Supplementary Figs. 35 and 36).
Our Revision: Relevant descriptions have been added in the main text as below.
Pages 10-11 in the main text: The gating voltage was mainly affected by charge distribution changes induced by the dramatic conformational changes.
Page 14 in the main text: As the presence of DQ47 led to a fully folded (more condensed) conformation of LC46, this similar change pattern in current signals indicated that these small molecules also had the potential to cause extended-tocondensed conformational changes of LC46.

Response:
We thank the reviewer for the comments and suggestions. In comparison with absolute values of the current levels, we focus more on the relative relationship between different current states. Because the absolute value of currents will deviate with different devices and the testing environments, the relative value can better reflect the relationship between different species in the same testing system. In this work, in contrast to the LC46-inhibitor complexes, the LC46-DQ47 complex can form a more condensed and stable helical structure, making the global positive charge denser on the surface of the SiNW and leading to the formation of a lower conductivity state. The concentration dependence also indicates that, at high concentrations of DQ47, the signal changed from a bistable state with high conductivity to that with low conductivity. and DQ47 (Fig. R23). The averaged dwell times <t> were calculated from 1-sencond data. The figure illustrated the dynamic disorder nature of single-molecule events.
Response：We thank the reviewer for the suggestions about the PBS concentration. In order to fully detect the conformational change of LC46 and its interaction with other ligands, experiments were conducted with various concentrations of phosphatebuffered saline (PBS) to investigate the influence of ionic strength and provide an optimal signal-noise ratio. The experimental results are illustrated in Supplementary  Fig. 20, which is also given below, showing an optimal signal-noise ratio in 0.01× PBS with a Debye length of 7.5 nm. The signals were not obvious under high salt concentrations ( Supplementary Fig. 20). We agree that it is important to determine whether the protein exhibits the binding affinity in 0.01× PBS. We carried out SPR experiments under 0.01× PBS (0.0× PBS, 0.2% T20, 5% DMSO, pH7.4) and the kinetic analysis results are shown in Table R1 Fig. 19). In most periods, we observed a single conductivity state. We consider that this single conductivity state may not correspond to a specific conformation but a conformational ensemble with multiple conformations.
During the single current state, the disordered protein lied within an energy landscape without significant energy wells, and the transitions between conformations might be too fast (on the sub-μs to μs timescale) for us to capture.
The oscillatory signal aroused when the disordered protein transiently occupies a larger potential energy well on the energy landscape, resulting in a distinct conformational ensemble with a longer dwell time. We considered that there might be various sub-ensembles within the conformational ensemble, but the transition frequency between these sub-ensembles is faster than the sampling rate of our methods, requiring instruments with higher time resolution. We have also observed different conformational ensembles converging to the same current level, indicating distinct dynamic behaviors (as shown in Supplementary Fig. 24).
Furthermore, when interacting with partner molecules or ligands, specific conformational ensembles can be stabilized or induced by these molecules. These oscillation states represented several conformational ensembles rather than individual conformations. We considered that these conformational ensembles are different from the former conformational ensembles observed in the blank buffer.
Our Revision: We have added relevant revisions in the manuscript as below.
Page 7 in the main text: In the measurement of Myc-modified devices, the current trajectories showed a long-last single current stage with transient clustered signals (in 0.01× PBS, 5% DMSO, pH = 7.4) (Fig. 1c, Fig. 2a, and Supplementary Fig. 19c). Additionally, the authors have not provided an answer to the question of what changes, how many charges are involved, and what dynamic motions those charges undergo relative to the SiNW. Instead, the authors repeatedly claim that the signals and their changes originate from an "unknown" charge distribution resulting from "unknown" global conformational changes. The authors need to elaborate and provide more explanations regarding this issue. It would be possible to (1) calculate the protein's charge residues and total surface charges, (2) estimate their motions and distances from the SiNW during the protein's conformation, and (3) establish a connection between conformational changes of the protein or protein-partner complex and signal generation.

Response:
We thank the reviewer for the valuable comments. We agree that (1) Based on the charges of amino acid residues under physiological pH conditions, both the IDR LC46 and DQ47 employed in our study possess a net charge of +2e. However, as they do not exhibit stable or compact 3D structures in solution, it is difficult to accurately calculate surface charges.
(2) According to the references (J. Phys. Chem. B 2010, 114, 9, 3330, Nano Lett. 2013, we attempted to calculate the influence of selected structures on the C-terminus (linkage end) based on the Coulombic electrostatic potential model (Eq. R1). The conformations employed in our calculations are simulated based on the NMR results of LC46 (conformations generated from https://csrosetta.bmrb.io/submit) as shown in Fig. R2, where the purple conformation represents a partially-folded intermediate (apo state), and the green one represents the conformation in the Myc-Max complex. The results are shown in Table R2. The Myc-Max complex exhibits a higher Coulombic potential, occupying a lower conductive state in our study, suggesting that the Coulombic potential can provide some reference information. However, it should be noted that the peptides employed are smaller in size in comparison with SiNWs (as shown in Supplementary Fig. 18) and the sensing part of SiNWs cannot be approximated to a single point, which results in a much more complex condition between peptides and SiNWs. Coulombic potential method based on point charge model might be inadequate in this case. A more accurate theoretical model and rational designed experiments will be constructed in our further study. (3) Currently it is impossible for us to run molecular dynamics simulations to a time-scale that is comparable to the experimental time-scale due to huge computational time  Finally, we would like to thank all the referees very much for their patience, precious time and kind support.